1. Field of the Invention
The invention relates generally to assay methods for detection of biological molecules, viruses, and cells, and more specifically to solid-phase assay methods for detection of biological molecules, viruses, and cells in solution, based on active motion of analytes to and from probe molecules under action of electrostatic, magnetic, centrifugal (gravitation) or hydrodynamic forces.
2. Background Information
There are several principal limits in conventional bioassay methods: (i) diffusion limit for transport of analyte to immobilized probes, (ii) limits for the assay sensitivity due to limited affinity of probe molecules and, (iii) limits for the assay sensitivity due to unspecific binding of analyte molecules with surface, which we will further refer to as a background limit.
Diffusion Limit
In most practical cases of heterogeneous analysis assay time is limited by the diffusion of analyte molecules to the surface with immobilized probes. The problem was carefully analyzed upon development of the BIAcore plasmon resonance technique (Nieba et al., 1996; Myszka et al., 1998).
As an example, let us consider that probe molecules are immobilized on a substrate in a mono-layer with a surface density of Cs molecules per unit area. Let us also assume that probe molecules have infinitely large affinity with respect to the analyte molecules, so, that every molecule touching the surface binds to a probe molecule. Under these conditions time needed to saturate half of the probes on the surface will be described by the equation (Morozov & Morozova, (1992)):τ1/2≈Csδ/CoD  (1)
δ is the thickness of unstirred layer, D is the diffusion coefficient, Co is the analyte concentration in solution. The thickness of unstirred layer depends on the flow rate of liquid, V, and on the size of the surface, according to the following equation (Tunizkii, (1970)):δ=(vb/V)1/2(D/v)1/3  (2)                with v denoting the kinematic viscosity of liquid, and b denoting a characteristic size of the sensor element. Since the diffusion coefficient can only slightly be decreased by raising temperature the only way to enhance the diffusion-controlled reaction in such a passive heterogeneous system is to decrease the thickness of the unstirred layer. Intensive shaking of micro-titer plates, pressing solution through a narrow slot in the BIAcore apparatus are examples of solving the transport problem. However, these approaches cannot solve the problem completely: as seen from the Eqn. (2) the thickness of unstirred layer depends weakly on the flow rate. Thus, 100-fold increase in the rate of solution flow will accelerate the reaction only 10 times.        
As an example, let us consider a sensor with the size of 5×5 mm2 (b=5 mm) in a flow of analyte solution, V=5 cm/sec. For protein analyte molecules with the diffusion coefficient, D=5×10−10 m2/cek, moving in a water solution with viscosity, v=1×10−6 m2/sec, δ=(vb/V)1/2(D/v)1/3=25 μm. Assuming that sensor surface is covered by a monolayer of probe molecules, d=5 nm in diameter we estimate the density of probe molecules as Cs=1/Nad2. Na denotes the Avogadro number. Time needed to saturate half of all the probe from a solution with concentration, Co, can then be estimated as τ1/2=Csδ/2CoD=1.7×106/Co. Thus, even under such ideal conditions diffusion will limit the assay time of analyte solution with Co=1 nM to about 0.5 h. Therefore, there remains a need for methods for performing bioassays that decrease the time for performing the assay.
Affinity Limit.
In contrast to the assumption made in the example discussed above the real probe molecules (e.g., antibodies) in any practical system have a limited affinity, characterized by a dissociation constant, Kd, which is the ratio of the backward, kdiss, and the forward reaction rate, kass, as:Kd=kdiss/kass  (3)
Fraction of probe molecules, (AP)/P, occupied by analyte molecules bound at equilibrium from a solution with concentration, Co, is described by the equation:(AP)/P=Co/(Co+Kd)  (4)
Thus, to have half of the probe molecules occupied with the analyte its concentration in solution should be equal to Kd. Dissociation constants of antibodies vary between 10−7 M and 10−10 M and often present a limit for assay sensitivity.
Background Limit.
Background in bioassay often results from a weak unspecific interaction of analyte molecules with substrate. Though blocking the surface with proteins (BSA, dry milk and casein in immunoassay) and grafting inert polymers to surface (Chapman et al., 2000) considerably reduce the background, they never remove it completely. A radical method to reduce the background was suggested by Lee et al., (2000). They suggested applying a weak force to mechanically detach weakly bound analyte molecules from the surface. Since forces they applied were not enough to destroy specific interactions, only beads bound to the substrate surface via strong specific interactions remained. Therefore, there remains a need for methods for performing bioassays that provide reduced backgrounds.